Abstract
Experimental characterization of blood flow in living organisms is crucial for understanding the development and function of cardiovascular systems, but there has been no technique reported for snapshot imaging of thick samples in large volumes with high precision. We have combined computational microscopy and the diffraction-free, self-bending property of Airy-beams to track fluorescent beads with sub-micron precision through an extended axial range (up to 600 μm) within the flowing blood of 3 days post-fertilization (dpf) zebrafish embryos. The spatial trajectories of the tracer beads within flowing blood were recorded during transit through both cardinal and intersegmental vessels, and the trajectories were found to be consistent with the segmentation of the vasculature recorded using selective-plane illumination microscopy (SPIM). This method provides sufficiently precise spatial and temporal measurement of 3D blood flow that has the potential for directly probing key biomechanical quantities such as wall shear stress, as well as exploring the fluidic repercussions of cardiovascular diseases. Although we demonstrate the technique for blood flow, the ten-fold better enhancement in the depth range offers improvements in a wide range of applications of high-speed precision measurement of fluid flow, from microfluidics through measurement of cell dynamics to macroscopic aerosol characterizations.
Highlights
Localization microscopy has attracted enormous interest due to its ability to super-resolve the positions of small emitters in three dimensions with an uncertainty that is much less than the dimensions of the image of the emitter
We have recently demonstrated selective-plane-illumination microscopy in conjunction with micro particle image velocimetry (PIV) (SPIM-μPIV) and high-speed, heart-synchronised, multi-depth acquisition to enable 3D measurement of blood flow in zebrafish [24], but the plane-by-plane optically-sectioned acquisition precludes easy measurement of the axial component of the flow vectors
We report the first application of pupil-engineered localization microscopy to in vivo bloodflow characterization; in particular we use the Airy-CKM technique [3] to map blood flow within 3-dpf zebrafish with sub-100 nm precision and sub-1 ms temporal resolution
Summary
Localization microscopy has attracted enormous interest due to its ability to super-resolve the positions of small emitters in three dimensions with an uncertainty that is much less than the dimensions of the image of the emitter. Precise localization using conventional microscopy is limited by diffraction to thin planes of about a micron thick, which prevents localization of points in three dimensions over extended depth ranges This is important for characterization of blood flow in the smaller vasculature, for which typical dimensions range between 10 μm and 200 μm, high precision in 3D is required for accurate velocity measurement, and high frame rate is required to resolve pulsatile hemodynamics. We report the first application of pupil-engineered localization microscopy to in vivo bloodflow characterization; in particular we use the Airy-CKM technique [3] to map blood flow within 3-dpf zebrafish with sub-100 nm precision and sub-1 ms temporal resolution. The technique offers improvement to a wide range of additional biological problems involving 3D point localization in extended volumes; examples currently being researched include 3D traction-force microscopy for characterization of forces exerted by biological cells and super-resolution imaging of extended 3D samples
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